CN1148891A - Method of quantifying cholesterol in low-density of very-low-density lipoprotein - Google Patents

Method of quantifying cholesterol in low-density of very-low-density lipoprotein Download PDF

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CN1148891A
CN1148891A CN96190206A CN96190206A CN1148891A CN 1148891 A CN1148891 A CN 1148891A CN 96190206 A CN96190206 A CN 96190206A CN 96190206 A CN96190206 A CN 96190206A CN 1148891 A CN1148891 A CN 1148891A
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cholesterol
formula
compound
solubilizing agent
reagent
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柏原典彦
多多纳俊雄
首藤荣子
杉内博幸
入江徹美
上釜兼人
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Hitachi Chemical Diagnostics Systems Co Ltd
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Kyowa Medex Co Ltd
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Abstract

A method of quantifying cholesterol in low-density lipoprotein (LDL) or very-low-density lipoprotein (VLDL), which comprises determining the content of cholesterol in LDL or VLDL contained in the sample in the presence of a sugar compound and/or a protein solubilizer.

Description

Method for the quantification of cholesterol in low-density lipoproteins or very low-density lipoproteins
The present invention relates to a method for determining cholesterol in low-density lipoprotein (LDL) or very low-density lipoprotein (VLDL) (hereinafter referred to as LDL cholesterol and VLDL cholesterol), which is important in lipid metabolism in the field of clinical diagnosis.
LDL has an action of supplying cholesterol to peripheral cells, and is a direct factor causing various arteriosclerosis such as coronary arteriosclerosis, and the content of LDL in blood is known as an index of arteriosclerotic diseases. In addition, VLDL rich in Triglycerides (TG) is also involved in arteriosclerosis, which is attracting attention. Conventional methods for the determination of LDL cholesterol include ultracentrifugation, electrophoresis, and Algorithm, while methods for the determination of VLDL cholesterol include ultracentrifugation and electrophoresis. Ultracentrifugation can be used as a basic method for determination of the amount of cholesterol in LDL or VLDL by separating LDL or VLDL based on the difference in specific gravity using an ultracentrifuge for separation (Adr. lipid Res., vol.6, page 1, 1968). However, this method has disadvantages in terms of quantitativeness, simplicity, economy, etc. In the case of electrophoresis, separation is carried out using a support such as cellulose acetate membrane or agar gel, and cholesterol is quantified by an enzymatic reaction (clinical examination, Vol.29, p.1344, 1985). This method has problems in terms of simplicity, economy, and the like. In the case of the algorithm change, the LDL cholesterol content was calculated according to the following calculation formula (clin. chem., volume 18, page 499, 1972).
(LDL Cholesterol amount) (Total Cholesterol amount) - (HDL Cholesterol amount) - (triglyceride)/5
However, this method is limited by the TG content in serum, the type of hyperlipidemia, and the like, and therefore has problems in terms of simplicity, accuracy, handling of a plurality of samples, and the like. As described above, conventional methods for the determination of LDL cholesterol or VLDL cholesterol are not suitable for use in automatic analyzers which are widely used in the fields of processing a plurality of samples, rapid measurement, and clinical examinations. Further, according to the conventional quantitative method, human errors such as when the separated LDL is taken out with a quantitative pipette are likely to occur. However, even if a serum sample is directly added to a reagent containing cholesterol esterase and cholesterol oxidase without separating it into LDL or VLDL, there is no difference from the system for the determination of total cholesterol, and LDL cholesterol or VLDL cholesterol cannot be specifically determined.
The present inventors have found that when various lipoproteins such as high-density lipoprotein (HDL), LDL, VLDL and Chylomicron (CM) separated by ultracentrifugation are measured using a reagent system for measuring cholesterol in which a sugar compound and/or a protein solubilizing agent are present, the reactivity with the various lipoproteins can be differentiated by the combination of the sugar compound and/or the protein solubilizing agent, and as a result, the reactivity with cholesterol in HDL, LDL cholesterol, VLDL cholesterol and CM can be differentiated, and thus the present invention has been completed.
The present invention relates to a method for the determination of LDL cholesterol or VLDL cholesterol, which is characterized by measuring the LDL cholesterol or VLDL cholesterol in a sample in the presence of a sugar compound and/or a protein solubilizer. In the above measurement, a divalent metal atom salt may be added at the same time in order to further improve the specificity.
Further, the present invention provides a reagent for the determination of cholesterol in LDL or VLDL, which comprises a sugar compound and/or a protein solubilizing agent as a component, or a reagent for the determination of cholesterol in LDL or VLDL, which comprises a combination of a sugar compound and a protein solubilizing agent.
As the sugar compound, a glucose derivative is preferably used, and examples of the glucose derivative include compounds represented by the general formulae (I) and (II) in which,
the general formula (I) is:
Figure A9619020600071
[ in the formula, R1、R2And R3May be the same or differentAnd represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkanoyl group, SO3M2(in the formula, M2Represents a hydrogen atom or a metal atom), - (glucosyl group)p-H (wherein p represents 1 or 2) or- (maltosyl)q-H (wherein q represents 1 or 2); r4And R5May be the same or different and represents a hydrogen atom, a metal atom or SO3M3(in the formula, M3Represents a hydrogen atom or a metal atom); m represents 6-8]
The general formula (II) is:
[ in the formula, R6、R7And R8May be the same or different and represents hydrogen or SO3M4(in the formula, M4Represents a hydrogen atom or a metal atom); r9Represents a hydrogen atom, OM5(in the formula, M5Representing a hydrogen atom or a metal atom) or OSO3M6(in the formula, M6Represents a hydrogen atom or a metal atom); r10Represents a hydrogen atom, a metal atom or SO3M7(in the formula, M7Represents a hydrogen atom or a metal atom); n represents an integer of 4 to 8000].
As the protein solubilizing agent, it is preferable to use a compound represented by the general formula (III), (IV) or (V) in which,
the general formula (III) is:
R11(C2H4O)a-(C3H6O)bR12(III)
[ in the formula, a and b represent integers of 0 to 200; r11Represents R20-X-O- (wherein, R20Represents an alkyl or alkenyl group; x represents a single bond or CO), or H- (CH)2CH2O)c-N(R21) - (wherein, c represents an integer of 1 to 200); r21Represents an alkyl group or a bonded alkenyl group); r12Is represented by C2H4COOR22、C3H6COOR23、C2H4CH(COOR24)2Or C2H4CH(COOR25)(COOR26) (in the formula, R22、R23、R24、R25And R26Identical or different, represents a hydrogen atom, a metal atom, an alkyl or alkenyl group) but a and b cannot both be 0, although both components may be present in any ratio]
The general formula (IV) is:
(in the formula, R13、R14、R15、R16、R17And R18The same or different, represent an alkanoyl group)
The general formula V is:
R19-Y-SO3M1(V)
[ in the formula, R19Represents an alkyl group, an alkenyl group or a substituted or unsubstituted aryl group; y represents a single bond,
Figure A9619020600092
-O-、-CH(R27) - (in the formula, R27Represents an alkyl or alkenyl), -CH2CH(OH)(CH2)d- (wherein d represents an integer of 1 to 22), -CH ═ CH (CH)2)e- (wherein e represents an integer of 1 to 22), -OCOCH (CH)2COOR28) - (in the formula, R28Represents an alkyl or alkenyl group) or a mixture thereof; m1Represents hydrogen or a metal atom].
The compounds represented by the general formula (I) to the general formula (V) are hereinafter referred to as compound (I) to compound (V), respectively.
In the definitions of the groups of the general formula (I) to the general formula (V), examples of the alkyl group and the alkyl moiety of the alkanoyl group which may be straight-chain or branched-chain alkyl group having 1 to 22 carbon atoms include: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, decyl, pentadecyl, eicosyl, docosyl and the like; the alkenyl group means a straight-chain or branched alkenyl group having 2 to 22 carbon atoms, and examples thereof include: vinyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, decenyl, pentadecenyl, eicosenyl, docosenyl, and the like. Said aryl group represents phenyl or naphthyl; examples of the metal atom include lithium, sodium, and potassium.
Examples of the substituent for the substituted alkyl group and the substituted alkanoyl group include: hydroxyl, carboxyl, sulfo, and the like. Examples of the substituent for the substituted aryl group include an alkyl group, and the alkyl group has the same definition as the above alkyl group.
Examples of the sugar compound include α -cyclodextrin, β -cyclodextrin, γ -cyclodextrin, dimethyl- β 0-cyclodextrin, trimethyl- β 1-cyclodextrin, hydroxyethyl- β 3-cyclodextrin, 2-hydroxypropyl- β 2-cyclodextrin, 2-hydroxypropyl- β -cyclodextrin, carboxymethyl- β -cyclodextrin, glycosyl- β -cyclodextrin, maltosyl- α -cyclodextrin, maltosyl- β -cyclodextrin, partial (パ - シヤリ) -methyl- β -cyclodextrin, α -cyclodextrin sulfate, β -cyclodextrin sulfate, and the like.
As the protein solubilizing agent, a surfactant such as compound (III), compound (IV) or compound (V) is preferably used, and a nonionic surfactant and an anionic surfactant are particularly preferably used. Examples of the nonionic surfactant include: polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, polyoxyethylene behenyl ether, polyoxyethylene monolaurate, polyoxyethylene monostearate, polyoxyethylene monooleate, polyoxyethylene lauramide, polyoxyethylene stearamide, sucrose fatty acid ester, and the like; examples of the anionic surfactant include: sodium dodecyl benzene sulfonate, sodium lauryl sulfate, sodium higher alcohol sulfate, etc.
Examples of divalent metal atom salts include: 0.01-20mM magnesium salt, calcium salt, manganese salt, nickel salt, cobalt salt, etc., preferably 0.01-20mM magnesium salt.
The present invention is characterized in that a sugar compound and/or a protein solubilizing agent is allowed to coexist with a reagent system for measuring cholesterolThe preparation itself can be prepared in accordance with a general method based on the following reaction principle. The dye source and the measurement wavelength are not limited.
Pigment + 5H2O(λmax555nm) EMSE: N-ethyl-N- (3-methylphenyl) -N' -succinylethylenediamine
NAD(P)H＋H+(λmax＝340nm)
For example, as the dye source, a combination of 4-aminoantipyrine and phenols such as phenol, 4-chlorophenol, m-cresol, 3-hydroxy-2, 4, 6-triiodobenzoic acid (HTIB) is generally used, and in addition thereto, 4-aminoantipyrine can be used in combination with the following compounds known to be used as Trinder's reagent (Dojindo laboratories, 19 th edition, 1994): n-sulfopropylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) m-Toluidine (TOOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethylaniline (MAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-Dimethoxyaniline (DAOS), N-ethyl-N-sulfopropyl-m-Toluidine (TOPS), N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethoxyaniline (HDAOS), N-dimethyl-m-toluidine, N-disulfopropyl-3, 5-dimethoxyaniline, N-ethyl-N-sulfopropyl-m-toluidine, Anilines such as N-ethyl-N-sulfopropylaniline, N-ethyl-N-sulfopropyl-3, 5-dimethoxyaniline, N-ethyl-N-sulfopropyl-3, 5-dimethylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) m-anisidine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline, and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethoxyaniline, or N-ethyl-N- (3-methylphenyl) -N' -succinylethylenediamine (EMSE), N-ethyl-N- (3-methylphenyl) -N' -acetylethylenediamine, and the like. As a highly sensitive dye source, 10- (N-methylcarbamoyl) -3, 7-bis (dimethylamino) phenothiazine (MCDP) shown in Japanese patent publication No. 60-33479, bis [ 3-bis (4-chlorophenyl) methyl-4-dimethylaminophenyl]amine (BCMA) shown in Japanese patent publication No. 4-27839, a dye source shown in Japanese patent application laid-open No. 62-296, or the like may be used, and 4-aminoantipyrin or the Trinder's reagent described above may be used in combination. The concentration of the pigment source is preferably 0.01 to 10mg/ml, which is limited by the solubility.
Examples of cholesterol ester hydrolase, cholesterol oxidase or cholesterol dehydrogenase include cholesterol esterase or lipoprotein lipase obtained from some microorganisms or animals generally commercially available, which have the ability to hydrolyze cholesterol ester, cholesterol oxidase obtained from some microorganisms or animals which oxidize cholesterol to generate hydrogen peroxide, cholesterol dehydrogenase obtained from microorganisms or animals, and the like. Further, enzymes produced by extracting genes from these enzymes according to genetic engineering procedures and introducing the genes into other microorganisms, modified bodies obtained by chemically modifying these enzymes, enzymes produced by modifying these genes, modified bodies obtained by chemically modifying these enzymes, and the like are also suitably used.
Examples of the reagent (chemical modifier) for modifying the enzyme include: a compound produced by bonding a group capable of bonding to an amino group to polyethylene glycol { for example, Sunbright VFM 4101 [ manufactured by japan fat and oil (ltd.)]produced by bonding a group capable of bonding to an amino group such as N-hydroxysuccinimide group to polyethylene glycol }, Sunbright AKM series having a polyethylene glycol structure and an acid anhydride structure, the same ADM series, and the same ACM series [ each manufactured by japan fat and oil (ltd.): the theory of chemical engineering, volume 20, No. 3, page 459, 1994, a compound formed by bonding a group capable of bonding to an amino group to a copolymer of polyethylene glycol and polypropylene glycol, a copolymer of polyethylene glycol monomethacrylmonomethylether and maleic anhydride, and the like. Further, polyurethane P4000activated as a polyurethane chemical modifier (Enzyme modification set, manufactured by ベ - リニガ - マンハイム), dextran T40 as a dextran chemical modifier, TCT-activated (same), 1, 3-propane sultone, and the like can also be used.
The method of reacting the chemical modifier with the enzyme will be described below, but the present invention is not limited to these examples. First, the enzyme is dissolved in a buffer solution such as phosphate buffer solution having a pH of 8 or more, and Sunbright, for example, in an amount of 0.01 to 500 times by mole is added thereto at 0 to 50 ℃ and stirred for 5 minutes to 24 hours. The reaction solution may be used as it is or after removing low molecular substances by an ultrafiltration membrane as necessary. The cholesterol ester hydrolase, cholesterol oxidase and cholesterol dehydrogenase are preferably used in an amount of 0.1-100. mu.g/ml.
The method of the present invention can be applied to a body fluid containing LDL or VLDL, such as blood or urine.
The following illustrates the quantitative method of the present invention.
Method 1
In practicing the present invention, a sugar compound solution and/or a protein solubilizing agent solution is first prepared. The sugar compound solution is prepared by dissolving the sugar compound in an appropriate buffer, for example, 50mM Tris-HCl buffer (pH7.4), so that the concentration thereof during the reaction is, for example, 100mM or less, preferably 3 to 80 mM. The sugar compound may be present in the cholesterol measuring reagent in advance. The protein solubilizing agent solution is prepared by dissolving the protein solubilizing agent in an appropriate buffer, for example, 50mM Tris-HCl buffer (pH7.4), and allowing the solution to coexist with the cholesterol measuring reagent, so that the concentration of the solution during the reaction is, for example, 50g/l or less, preferably 0.1 to 20g/l or less. The reagent is prepared from a protein solubilizing agent solution which coexists with a sugar compound solution and/or a cholesterol measuring reagent (in the case where a protein solubilizing agent solution is not used, a sugar compound is previously coexisted in a cholesterol measuring reagent), and the resulting mixture is incubated at 20 to 50 ℃ and preferably 30 to 40 ℃ for about 5 minutes. The sample is added directly or after being diluted with water or saline as necessary, to the above reagent, and reacted for 5 to 30 minutes. After the reaction is finished, the reaction temperature is increased to 340 nm and 900nm, for example, 555nm in the case of single wavelength; the absorbance of the reaction solution was measured at a main wavelength of 600nm and a sub-wavelength of 700nm in the case of both wavelengths, and the cholesterol content was calculated therefrom (in the case of both wavelengths, the difference between the absorbances obtained at the respective wavelengths was calculated).
Each of HDL, LDL, VLDL and CM separated from serum by ultracentrifugation was used as a sample, and the cholesterol content was measured using the above-mentioned reagent. As a result, it was confirmed that the reactivity with various lipoproteins can be differentiated by the combination of the sugar compound and the protein solubilizing agent by differentiating the reactivity of HDL cholesterol, LDL cholesterol, VLDL cholesterol, and CM cholesterol.
When 50mM of sugarcompound was combined with 5g/l of polyoxyethylene monolaurate, a protein solubilizer, and co-present in the cholesterol assay reagent (unmodified), the difference in reactivity of various lipoproteins is shown in Table 1.
TABLE 1
Sugar compounds HDL LDL VLDL CM
α Cyclodextrin + + + + +
β Cyclodextrin + + + + +
Gamma-cyclodextrin + + + + + +
Dimethyl- β -cyclodextrin - + + + + + + + + +
Trimethyl- β -cyclodextrin - + + + + +
Hydroxyethyl- β -cyclodextrin - + + + +
2-hydroxypropyl- α -cyclodextrin + + + + + + +
2-hydroxypropyl- β -cyclodextrin - + + + + + +
Carboxymethyl- β -cyclodextrin + + + + + + +
Glucosyl- β -cyclodextrin + + + + + + +
Maltosyl- α -cyclodextrin + + + + + + +
Maltosyl- β -cyclodextrin + + + + +
Partial methyl- β -cyclodextrin + + + + +
α Cyclodextrin sulphate + + + + +
β Cyclodextrin sulphate + + + + +
-, + + + + +, respectively represent the reactivity strength, and the sequence is-<+ + +.
When the sugar compound trimethyl- β -cyclodextrin 5mM was combined with a protein solubilizing agent 5g/l and co-present in the cholesterol measuring reagent (unmodified), the difference in reactivity of the various lipoproteins is shown in Table 2.
TABLE 2
Protein solubilizer HDL LDL VLDL CM
Polyoxyethylene eicosyl ether - + + + + + + + + +
Polyoxyethylene cetyl ether + + + + +
Polyoxyethylene stearyl ether + + + + +
Polyoxyethylene octadecenyl ether + + + + +
Polyoxyethylene behenyl ether + + + + +
Polyoxyethylene monolaurate - + + + + +
Polyoxyethylene monostearate - + + + +
Polyoxyethylene monooleate - + + + +
Polyoxyethylene laurylamides - + + + +
Polyoxyethylene stearamide - + + + +
Sucrose fatty acid ester + + + + + +
Sodium dodecyl benzene sulfonate - + + + +
Sodium n-dodecylbenzenesulfonate - + + + + + +
Sodium dodecyl sulfate - + + + + + +
Higher alcohol sodium sulfate + + + + +
-, + + + + respectively represent the reaction intensity, and the sequence is-<+ + +.
Method 2
The sugar compound solution is prepared by dissolving the sugar compound in an appropriate buffer, for example, 50mM Tris-HCl buffer (pH7.4), so that the concentration of the sugar compound during the reaction is, for example, 100mM or less, preferably 3 to 80 mM. The protein solubilizing agent solution is prepared by dissolving the protein solubilizing agent in an appropriate buffer, for example, 50mM Tris-HCl buffer (pH7.4), so that the concentration thereof during the reaction is, for example, 50g/l or less, preferably 0.1 to 20 g/l. The sample is added directly or, if necessary, after dilution with water or physiological saline, to a solution of a sugar compound and/or a protein solubilizing agent heated in advance to 20 to 50 ℃, preferably 30 to 40 ℃, for example, 37 ℃, for example, heated at 37 ℃ for 5 minutes, and the absorbance at 555nm of the obtained solution is measured after 5 minutes (E1). Then, a cholesterol measurement reagent previously heated to 20 to 50 ℃, preferably 30 to 40 ℃, for example 37 ℃ is added thereto, and the mixture is stirred, and after 5 minutes, the absorbance [ E2 (value after concentration correction)]is measured at the same wavelength. The same procedure was carried out using a standard solution containing cholesterol at a known concentration, and the cholesterol content was calculated from a comparison of the (E2-E1) values.
The following describes the scheme of the present invention according to examples.
Best mode for carrying out the invention
Example 1
Comparison of this method for the direct quantification of LDL cholesterol with agarose electrophoresis (clinical examination, Vol.29, p.1344, 1985).
Reagent composition of the method
A first reagent
Trimethyl- β -Cyclodextrin 5mM
5g/l polyoxyethylene monolaurate
EMSE1.1mM
Tris buffer (pH7.0) 30mM
Second reagent
Cholesterol esterase (unmodified) 1.0U/ml
Cholesterol oxidase (unmodified) 5.0U/ml
Peroxidase 25U/ml
2.2mM of 4-chloro-antipyrine
Tris buffer (pH7.0) 30mM
According to the method, 50. mu.l of a serum sample is first added to 2.25ml of a first reagent previously heated to 37 ℃ and heated at 37 ℃ for 5 minutes, and the absorbance of the resulting solution at 555nm is measured after 5 minutes (E1). Then, 0.75ml of a second reagent previously heated to 37 ℃ was added thereto, and after 5 minutes, the absorbance at the same wavelength [ E2 (corrected concentration value)]was measured. The same procedure was carried out using a standard solution having a cholesterol concentration of 200mg/dl, and the LDL cholesterol content was calculated by comparing the values (E2-E1).
The lipoprotein fraction on the support after electrophoresis was subjected to enzymatic staining for cholesterol according to the agarose electrophoresis method, and the LDL cholesterol content was quantitatively determined by a densitometer (manufactured by クリニスキヤン K.).
The results obtained are shown in table 3.
TABLE 3
Sample (I) LDL Cholesterol concentration (mg/dl)
Method of the invention Electrophoresis method
1 2 3 4 5 6 7 8 9 10 62 85 77 148 122 156 150 133 133 140 55 81 72 138 116 151 139 121 123 129
As shown in Table 3, it was shown that the results obtained by the method of the present invention have a good correlation with the results obtained by the electrophoresis method.
Example 2
The same procedure as in the method of the present invention of example 1 was carried out except that the sugar compound used in the first reagent and the protein solubilizing agent were changed in combination, and 20 specimens of the serum samples were measured to obtain the correlation coefficient with respect to the agarose electrophoresis method.
Composition of the first reagent
A. Trimethyl- β -Cyclodextrin 5mM
5g/l polyoxyethylene monolaurate
EMSE 1.1mM
Tris buffer (pH7.0) 30mM
B. Trimethyl- β -Cyclodextrin 5mM
5g/l sodium dodecyl benzene sulfonate
EMSE 1.1mM
Tris buffer (pH7.0) 30mM
C. Dimethyl- β -Cyclodextrin 5mM
5g/l polyoxyethylene monolaurate
EMSE 1.1mM
Tris buffer (pH7.0) 30mM
D. Dimethyl- β -Cyclodextrin 5mM
5g/l sodium dodecyl benzene sulfonate
EMSE 1.1mM
Tris buffer (pH7.0) 30mM
According to this method, measurement was carried out using a Hitachi 7070 automatic analyzer. The measurement conditions are as follows.
Sample amount: 4 μ l
A firstreagent: 300 μ l
A second reagent: 100 μ l
Measurement of wavelength dominant wavelength: 600 nm; secondary wavelength: the results obtained at 700nm are shown in Table 4. TABLE 4
A first reagent Correlation coefficient
A. 0.9324
B. 0.8227
C. 0.8523
D. 0.7876
As shown in Table 4, it was revealed that the results obtained by the method of the present invention correlated well with the results obtained by the electrophoresis method.
Example 3
The procedure of example 2 was repeated except that a metal atom salt was added to B and D of example 2, and 20 samples of the serum samples were measured to obtain a correlation coefficient by the agarose electrophoresis method.
Composition of the first reagent
E. Trimethyl- β -Cyclodextrin 5mM
5g/l sodium dodecyl benzene sulfonate
MgCl2·6H2O 6mg/ml
EMSE 1.1mM
Tris buffer (pH7.0) 30mM
F. Dimethyl- β -Cyclodextrin 5mM
5g/l sodium dodecyl benzene sulfonate
MgCl2·6H2O 6mg/l
EMSE 1.1mM
Tris buffer (pH7.0) 30mM
The results are shown in Table 5.
TABLE 5
A first reagent Correlation coefficient
E. 0.9302
F. 0.9298
As shown in Table 5, it was revealed that the results obtained by the method of the present invention had a good correlation with the results obtained by the electrophoresis method.
Example 4
The procedure for directly determining VLDL cholesterol was compared with the procedure of example 1 using agarose electrophoresis (clinical examination, Vol.29, p.1344, 1985).
Reagent composition of the method
A first reagent
2-hydroxypropyl- β -Cyclodextrin 5mM
5g/l polyoxyethylene dodecyl ether
EMSE 1.1mM
Tris buffer (pH7.0) 30mM
Second reagent
Modified cholesterol esterase 1.0U/ml
Modified cholesterol oxidase 5.0U/ml
Peroxidase 25U/ml
2.2mM 4-aminoantipyrine
Tris buffer (pH7.0) 30mM
The modified cholesterol esterase and the modified cholesterol oxidase were prepared by dissolving cholesterol esterase or cholesterol oxidase in 20mM phosphate buffer (pH8) (10mg/ml), cooling at 5 ℃ and adding 20 times the molar amount of Sunbright4001 (Japanese fat and oil) thereto to dissolve them, reacting at 5 ℃ for 4 hours, and modifying with polyethylene glycol to obtain a reaction solution which was used as it was (molecular weight of polyethylene glycol portion 6000).
The results are shown in Table 6.
TABLE 6
Sample (I) LDL Cholesterol concentration (mg/dl)
Method of the invention Electrophoresis method
1 2 3 4 5 6 7 8 24 29 17 19 12 25 46 44 19 22 15 23 15 23 49 39
9 10 33 31 27 34
As shown in Table 6, it was revealed that the results obtained by the method of the present invention had a good correlation with the results obtained by the electrophoresis method.
Possibility of industrial utilization
According to the present invention, a method for the determination of LDL cholesterol or VLDL cholesterol can be provided which is simple and convenient without requiring a complicated fractionation operation and is suitable for use in an automatic analyzer.

Claims (21)

1. A method for the determination of cholesterol in low-density lipoprotein (LDL) or very low-density lipoprotein (VLDL), characterized in that the cholesterol content in LDL or VLDL in a sample is measured in the presence of a sugar compound and/or a protein solubilizing agent.
A method for the determination of cholesterol in LDL, characterized by measuring the cholesterol content in LDL in a sample in the presence of a sugar compound and/or a protein solubilizing agent.
A method for the determination of cholesterol in VLDL, which is characterized in that the cholesterol content in VLDL in a sample is measured in the presence of a sugar compound and/or a protein solubilizing agent.
4. A quantitative method as defined in claim 1 to 3, wherein said sugar compound is a glucose derivative.
5. A quantitative method according to claim 4, wherein the sugar compound is a compound represented by the following general formula (I) or general formula (II):
Figure A9619020600021
[ in the formula, R1、R2And R3May be the same or different and represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkanoyl group, SO3M2(in the formula, M2Represents a hydrogen atom or a metal atom), - (glucosyl) p-H (wherein p represents 1 or 2) or- (maltosyl)q-H (wherein q represents 1 or 2); r4And R5May be the same or different and represents a hydrogen atom, a metal atom or SO3M3(in the formula, M3Represents a hydrogen atom or a metal atom); m represents 6-8]
[ hereinafter referred to as compound (I), and the same holds true for the other compounds of the general formula (I);
said general formula (II) is:
Figure A9619020600031
[ in the formula, R6、R7And R8May be the same or different and represents hydrogen or SO3M4(in the formula, M4Represents a hydrogen atom or a metal atom); r9Represents a hydrogen atom, OM5(in the formula, M5Representing a hydrogen atom or a metal atom) or OSO3M6(in the formula, M6Represents a hydrogen atom or a metal atom); r10Represents a hydrogen atom, a metal atom or SO3M7(in the formula, M7Represents a hydrogen atom or a metal atom); n represents an integer of 4 to 8000].
6. A quantitative method as defined in claim 5, wherein said sugar compound is a cyclodextrin derivative.
7. The quantitative method according to claim 1 to 6, wherein the protein solubilizing agent is a compound represented by the general formula (III), (IV) or (V),
the general formula (III) is:
R11(C2H4O)a-(C3H6O)bR12(III)
[ in the formula, a and b represent integers of 0 to 200; r11Represents R20-X-O- (wherein, R20Represents an alkyl or alkenyl group; x represents a single bond or CO), or H- (CH)2CH2O)c-N(R21) - (wherein, c represents an integer of 1 to 200); r21Represents an alkyl group or a bonded alkenyl group); r12Is represented by C2H4COOR22、C3H6COOR23、C2H4CH(COOR24)2Or C2H4CH(COOR25)(COOR26) (in the formula, R22、R23、R24、R25And R26Identical or different, represents a hydrogen atom, a metal atom, an alkyl or alkenyl group) but a and b cannot both be 0, although both components may be present in any ratio]
The general formula (IV) is:
Figure A9619020600032
(in the formula, R13、R14、R15、R16And R18The same or different, represent an alkanoyl group)
The general formula V is:
R19-Y-SO3M1(V)
[ in the formula, R19Represents an alkyl group, an alkenyl group or a substituted or unsubstituted aryl group; y represents a single bond,
Figure A9619020600041
-O-、-CH(R27) - (in the formula, R27Represents an alkyl or alkenyl), -CH2CH(OH)(CH2)d- (wherein d represents an integer of 1 to 22), -CH ═ CH (CH)2)e- (wherein e represents an integer of 1 to 22), -OCOCH (CH)2COOR28) - (in the formula, R28Represents an alkyl or alkenyl group) or a mixture thereof; m1Represents a hydrogen atom or a metal atom].
8. A quantitative method according to claim 7, wherein the protein solubilizing agent is a nonionic surfactant or an anionic surfactant.
9. The quantitative method of any one of claims 1 to 8, wherein a divalent metal atom salt is present in the determination of cholesterol content.
10. The method according to any one of claims 1 to 9, wherein the cholesterol ester hydrolase is reacted with cholesterol oxidase or cholesterol dehydrogenase in a sample, and the hydrogen peroxide or reduced coenzyme generated by the reaction is quantitatively analyzed to determine the cholesterol content, and the cholesterol ester hydrolase, cholesterol oxidase or cholesterol dehydrogenase used in the method is a chemically modified or unmodified cholesterol esterase, a chemically modified or unmodified cholesterol oxidase or a chemically modified or unmodified cholesterol dehydrogenase.
11. A reagent for the determination of cholesterol in LDL or VLDL, which comprises a sugar compound and/or a protein solubilizing agent as a component.
12. A reagent for quantifying cholesterol in LDL, which comprises a sugar compound and/or a protein solubilizing agent as an ingredient.
13. A reagent for the determination of cholesterol in VLDL, which comprises a sugar compound and/or a protein solubilizing agent as a component.
14. A reagent for the quantification of cholesterol in LDL or VLDL, which comprises a sugar compound and a protein solubilizing agent.
15. A reagent for quantifying cholesterol in LDL, which comprises a sugar compound and a protein-solubilizing agent.
16. A reagent for the quantification of cholesterol in VLDL, which comprises a sugar compound and a protein solubilizing agent.
17. The quantifying reagent according to any one of claims 11 to 16, wherein the sugar compound is a glucose derivative.
18. The quantifying reagent according to claim 17,wherein the sugar compound is compound (I) or compound (II).
19. The quantifying reagent of claim 18, wherein the sugar compound is a cyclodextrin derivative.
20. The quantification reagent according to any one of claims 11 to 19, wherein the protein solubilizing agent is compound (III), compound (IV) or compound (V).
21. The quantification reagent of claim 20, wherein the protein solubilizing agent is a nonionic surfactant or an anionic surfactant.
CN96190206A 1995-03-20 1996-03-15 Method of quantifying cholesterol in low-density of very-low-density lipoprotein Pending CN1148891A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1313620C (en) * 2000-11-14 2007-05-02 第一化学药品株式会社 Method of lipid assay and reagent for use therein
CN111936861A (en) * 2018-03-30 2020-11-13 希森美康株式会社 Method and reagent for measuring lipoprotein absorption capacity

Families Citing this family (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3193634B2 (en) * 1996-05-29 2001-07-30 第一化学薬品株式会社 LDL cholesterol determination method
KR980010429A (en) * 1996-07-18 1998-04-30 다나까 모또아끼 Cholesterol Determination
EP0990904B1 (en) * 1997-04-14 2006-12-13 DENKA SEIKEN Co., Ltd. Method for quantitating cholesterol present in low density lipoproteins
US5814472A (en) * 1997-05-13 1998-09-29 Wako Pure Chemical Industries, Ltd. Measurement of LDL-cholesterol
JP3441993B2 (en) * 1999-01-27 2003-09-02 松下電器産業株式会社 Cholesterol sensor
DE60018039T2 (en) * 1999-11-22 2005-09-29 Matsushita Electric Industrial Co., Ltd., Kadoma SENSOR AND METHOD FOR MEASURING CHOLESTEROL
JP4456715B2 (en) * 2000-02-28 2010-04-28 協和メデックス株式会社 Method and reagent for measuring cholesterol in remnant-like lipoprotein
WO2001094619A1 (en) 2000-06-07 2001-12-13 International Reagents Corporation Method of analyzing components in biological samples
EP2194142A1 (en) * 2002-11-27 2010-06-09 Daiichi Pure Chemicals Co., Ltd. Method of measuring cholesterol in high density lipoproteins
EP1434054A1 (en) * 2002-12-25 2004-06-30 Matsushita Electric Industrial Co., Ltd. Biosensor for determining low density cholesterol
KR20060127111A (en) * 2004-01-29 2006-12-11 교와 메덱스 가부시키가이샤 Method, reagent, and kit for determining cholesterol in very-low-density lipoprotein remnant (vldl remnant)
US20060062688A1 (en) * 2004-02-03 2006-03-23 Polymer Technology Systems, Inc. Bodily fluid analysis system
US7625721B2 (en) 2004-02-03 2009-12-01 Polymer Technology Systems, Inc. Non-precipitating bodily fluid analysis system
US7435577B2 (en) * 2004-02-03 2008-10-14 Polymer Technology Systems, Inc. Direct measurement of chlolesterol from low density lipoprotein with test strip
US7491542B2 (en) * 2004-07-12 2009-02-17 Kim Scheuringer Test device for determining the concentration of LDL-cholesterol in a sample
US20060062690A1 (en) * 2004-08-17 2006-03-23 Polymer Technology Systems, Inc. Apparatus and method of manufacturing bodily fluid test strip
CA2636492A1 (en) * 2006-01-20 2007-07-26 Jimro Co., Ltd Method for measuring cholesterol in remnant-like lipoprotein
WO2008143159A1 (en) * 2007-05-18 2008-11-27 Kyowa Medex Co., Ltd. Method and kit for measurement of cholesterol in low-density lipoprotein
EP2116613B1 (en) 2007-09-05 2016-11-02 ARKRAY, Inc. Method and test piece for measuring low density lipoprotein (ldl) cholesterol
ES2744797T3 (en) 2008-02-29 2020-02-26 Univ Oxford Innovation Ltd Procedure for determining an adequate dose of statin
US9817001B2 (en) 2008-05-27 2017-11-14 Boston Heart Diagnostics Corporation Methods for determining LDL cholesterol treatment
US8470541B1 (en) 2008-09-27 2013-06-25 Boston Heart Diagnostics Corporation Methods for separation and immuno-detection of biomolecules, and apparatus related thereto
WO2010148130A1 (en) * 2009-06-17 2010-12-23 Maine Standards Company, Llc Method for measuring lipoprotein-specific apolipoproteins
US20140147869A1 (en) 2010-10-29 2014-05-29 Arkray, Inc. Method and Kit for Measuring Cholesterol in Low-Density Lipoproteins
US8765377B2 (en) 2011-10-13 2014-07-01 Boston Heart Diagnostics Corporation Compositions and methods for treating and preventing coronary heart disease
KR102072251B1 (en) 2011-11-11 2020-01-31 엑시스-시일드 에이에스 Blood sample assay method
JP6091158B2 (en) * 2012-10-23 2017-03-08 デンカ生研株式会社 Method to increase sensitivity of immunoassay system by pretreatment of urine with denaturant
US9828624B2 (en) 2013-07-24 2017-11-28 Boston Heart Diagnostics Corporation Driving patient compliance with therapy
US9739790B2 (en) 2014-11-17 2017-08-22 Boston Heart Diagnostic Corporation Cardiovascular disease risk assessment

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2513766B1 (en) * 1981-09-28 1985-09-13 Biomerieux Sa METHODS AND REAGENTS FOR SELECTIVE SEPARATION OF LOW DENSITY LDL LIPOPROTEINS AND FOR QUANTIFICATION OF THEIR COMPONENTS
DE3208253A1 (en) * 1982-03-08 1983-09-15 Boehringer Mannheim Gmbh, 6800 Mannheim METHOD FOR SPECIFIC DETERMINATION OF THE CHOLESTERIN OF THE LDL FRACTION IN SERUM
EP0355864A3 (en) * 1984-03-15 1991-09-18 Wako Pure Chemical Industries, Ltd. Method of quantitatively measuring an oxidative substance by using triphenyl methane type leuco compounds as coloring matter
JPS60200167A (en) * 1984-03-24 1985-10-09 Toyobo Co Ltd Quantitative analysis of hydrogen peroxide by chemiluminescent method
US4626511A (en) * 1985-05-13 1986-12-02 Wayne State University Composition for reducing turbidity in samples of biological fluids
DE3678356D1 (en) * 1985-08-06 1991-05-02 Kao Corp LIQUID SHAMPOO COMPOSITION.
DE3533288A1 (en) * 1985-09-18 1987-03-26 Boehringer Mannheim Gmbh METHOD AND REAGENT FOR SPECIFIC DETERMINATION OF HDL CHOLESTEROL IN SERUM
DE3636851A1 (en) * 1986-10-29 1988-05-11 Boehringer Mannheim Gmbh METHOD AND REAGENT FOR SPECIFIC DETERMINATION OF THE CHOLESTERIN OF THE HDL FACTION
US5032503A (en) * 1988-06-22 1991-07-16 Microgenics Corporation Liquid single reagent for air enzyme complementation assay
US5286626A (en) * 1991-12-13 1994-02-15 Actimed Laboratories, Inc. Process and apparatus for direct determination of low density lipoprotein
JP2653755B2 (en) * 1994-03-08 1997-09-17 協和メデックス株式会社 Determination of cholesterol in high density lipoprotein
JP2600065B2 (en) * 1994-03-08 1997-04-16 協和メデックス株式会社 Determination of cholesterol in high density lipoprotein
JP3107492B2 (en) * 1994-04-05 2000-11-06 国際試薬株式会社 Method for quantification of components in lipoprotein fraction
EP1650569A3 (en) * 1995-03-16 2006-05-17 Kyowa Medex Co., Ltd. Method for determination of cholesterol in low-density lipoprotein

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1313620C (en) * 2000-11-14 2007-05-02 第一化学药品株式会社 Method of lipid assay and reagent for use therein
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CN111936861B (en) * 2018-03-30 2023-09-01 希森美康株式会社 Method and reagent for measuring absorption capacity of lipoprotein

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